Flaviviruses are a significant cause of human morbidity and mortality with arthropod-borne transmission of these agents resulting in thousands of deaths per annum. Classical vector control has been the primary strategy to combat mosquito-borne diseases;however, population expansion of mosquito vectors, coupled with a lack of anti-viral therapeutics serves to emphasize the urgent need for vaccine development. The yellow fever (YF) 17D vaccine has been regarded as one of the safest and most efficacious vaccines ever developed and as such has become a platform for the development of live attenuated flavivirus vaccines for heterologous flaviviruses. Since these attenuated viruses are replication competent, inducing viremia in vertebrates and capable of mild arthropod infectivity caution has been recommended in their use due to the theoretical potential for recombination between vaccine and wild type strains. The purpose of this proposal is to evaluate the safety of these 17D based viruses with specific regard to the potential for recombination. We hypothesize that live attenuated flavivirus vaccine recombination is likely an extremely inefficient process due to attenuated in vivo vaccine replication kinetics and stringent molecular constraints required for generation of a viable recombinant. Experiments have been designed to evaluate the potential for sequence tagged 17D viruses to undergo recombination in cell culture and to assess the potential for recombination to occur in the mosquito vector. The proposed studies are highly significant to the development of vaccines and improvement of public health, in flavivirus endemic regions, because they directly address this major safety concern, by evaluating the potential for emergence of flavivirus vaccine recombinants. PUBLIC HEALTH RELEVANCE: Project Narrative: Mosquito-borne viruses are a major cause of human illness with arthropod-borne transmission of these agents resulting in thousands of deaths annually. Live attenuated vaccines are an attractive strategy for control of these agents;however concerns have been raised about the potential for these vaccine viruses to evolve in nature. The studies contained in this proposal are highly significant to the development of vaccines and improvement of public health in regions of mosquito-borne virus transmission, because they directly address this safety concern.